The rapid advances made in the last decade in gene transfer methods have created the opportunity for developing gene therapy for human neurological disease. One goal of therapy is to slow neuron loss, through the expression of neuroprotective genes. The development of gene therapies for chronic diseases critically depends on not only long-term expression of a therapeutic gene product, but also the development of non-toxic gene delivery systems. This project specifically focuses on refining herpes simplex virus (HSV) amplicon vectors, plasmid-based vectors that require a helper virus for packaging. Cytotoxicity associated with amplicon vector stocks appear to be largely a function of helper virus expression of HSV immediate early (IE) gene products. We plan to utilize two approaches to produce amplicon stocks which exhibit reduced cytotoxicity: One includes the use of a three component regulatory packaging system that utilizes tetracycline-dependent expression of IE gene products to increase amplicon to helper titer; the second approach includes the use of Cre recombinase mutants to derive packaging cell lines that cleave loxP-containing helper virus, resulting in reduced helper virus titers and in effect, decreased cytotoxicity in amplicon vector stocks.